Method for obtaining ngn3-expressing cells and insulin producing-beta cells

ABSTRACT

The present invention relates to a method for obtaining Ngn3-expressing cells and insulin producing-beta cells by contacting a Pdx1-expressing pancreas explant with an amount of at least one histone deacetylase inhibitor (HDACi). The inventive methods have the advantage of being simple and quick, and of providing large amounts of Ngn3-expressing cells and insulin producing-beta cells, that are useful therapeutic tools. The invention also relates to a pharmaceutical composition for the treatment of diabetes which comprises an amount of at least one HDACi.

FIELD OF THE INVENTION

The invention relates to a method for obtaining a population of Ngn3-expressing cells or a population of insulin producing-beta cells by contacting a Pdx1-expressing pancreatic explant with an amount of at least one histone deacetylase inhibitor (HDACi). The invention also relates to a pharmaceutical composition for the treatment of diabetes which comprises an amount of at least one HDACi.

BACKGROUND OF THE INVENTION

Nowadays, more than 150 million people suffer from diabetes in the world. This disease is rising heavily and it is estimated than in the next 20 years, 300 million people could be affected. Insulinotherapy is the large-scale diabetes mellitus treatment. It consists in recurrent injections of insulin every day. The hope is to replace this heavy treatment, which is associated with side effects, by a definitive cure. In this goal, islets transplantation was tested according to the Edmonton protocol (66). However, 5 to 10 organ donors are required to transplant a single diabetic patient. Thus, one of the major problems limiting islet transplantation therapy is the lack of organ donors. An alternative source of beta cells (insulin producing-beta cells) is therefore required. Different approaches are being considered: xenografts, transdifferentiation of bone marrow, liver or intestine cells, as well as differentiation of embryonic or adult stem cells. Yet, despite of the intensive efforts which have been devoted to define an effective method for obtaining beta cells, none of these methods really are satisfying. Indeed, even if some of these strategies may be useful, each of these approaches has shown limitations (e.g., raised problems of rejection and potential transfer of viruses form animal to human with xenograft; important risk of carcinogenesis and difficulties to control in vitro some steps of development with embryonic stem cells; early stage of approaches based on adult stem cells, liver and intestine cells, and poor reproducibility encountered with bone marrow.)

Thus, there still remains, in the art, an ongoing need for an effective method for obtaining islet progenitors and more importantly insulin producing-beta cells useful in therapy, and more particularly in cell replacement in diabetes. With regard to this serious public health problem, well understanding the pancreatic development and processes controlling it is an essential and critical challenge. However, the mechanisms regulating cell fate choices during pancreatic development are still unclear.

Currently, there is known that the mature pancreas contains exocrine tissue composed of acinar cells that secrete digestive enzymes via a branched network of ductal cells into the intestine and endocrine islets that produce hormones such as insulin (β cells), glucagon (α cells), somatostatin (δ cells) and pancreatic polypeptide (PP cells). The pancreas originates from the dorsal and ventral regions of the foregut endoderm directly behind the stomach. Signals derived from adjacent mesodermal structures, notochord and dorsal aorta, (32, 36) and mesenchyme, which condenses around the underlying committed endoderm (4, 52) are involved in the control of pancreas development.

It has also been shown during pancreas development, transcription factors play critical roles in exocrine and endocrine differentiation. Indeed, studies in genetically engineered mice have identified a hierarchy of transcription factors regulating pancreatic specification, growth and differentiation. Thus, recent work has identified transcription factors that regulate pancreatic cell lineages (10, 29). The pancreas-committed endodermal region expresses the homeodomain factor Pdx1 (28, 49). Next, the basic helix-loop-helix factor Neurogenin3 (Ngn3) initiates the endocrine differentiation program in epithelial pancreatic progenitor cells. Indeed, Ngn3-deficient mice fail to generate any endocrine cells (18) and lineage tracing experiments have also provided direct evidence that Ngn3-expressing cells are islet progenitors (20). Subsequently, additional transcription factors determine the specific endocrine cell fate. Gain- and loss-of-function experiments are consistent with antagonistic roles for Pax4 and Arx in specifying endocrine sub-types (for β/δ or α/PP cells, respectively). Whereas Pax4-deficient mice display a selective loss of β- and δ-cells with a proportional increase in α-cells, Arx-deficient mice present the opposite phenotype (11, 54). Furtheimore, Arx and Pax4 display mutual transcriptional inhibition (8).

In other respects, transcriptional regulation in eukaryotes occurs within chromatin and is influenced by post-translational histone modifications (e.g. acetylation) involving histone deacetylases. Histone modifications play crucial roles in the transcriptional regulation of most eukaryotic genes and have been linked to cell differentiation control (in muscle and cardiac cells, for example (3, 43)). Acetylation or deacetylation of histone terminal domains can regulate gene expression. Thus, acetylation generally contributes to the formation of a transcriptionally competent environment by relaxing the chromatin structure, allowing transcription factors to access the target DNA. In contrast, histone deacetylation compacts chromatin and leads to transcriptional repression (19, 34). More precisely, histone acetyltransferases (HATS) and histone deacetylases (HDACs), respectively loosen or compact chromatin structures, regulate cell proliferation/differentiation in various tissues (6, 7, 35, 43, 47, 57, 59). Recent invalidation studies in mice revealed that the various HDACs are not functionally redundant. Thus, HDAC1 and -2 were shown to regulate cardiac development (57). HDAC5 and HDAC9 are involved in the heart's response to stress signals (6), HDAC4 in chondrocyte hypertrophy (59) and HDAC7 in maintenance of vascular integrity (7).

Small-molecule, called histone deacetylasc inhibitors (HDACi) are major tools for studying the connection between overall chromatin effects and cell lineage specification. Pharmacological inhibition of HDACs enables experimental manipulation and systematic analysis of chromatin remodelling (41). The effects of HDACi are selective (39, 58) and are thus often used to specifically inhibit HDACs (41, 45, 60). For example, valproic acid (VPA) preferentially targets class I HDACs (17), whereas trichostatin A (TSA) inhibits both class I and class II HDACs (Yoshida et al., 1990). HDACi were successfully used to demonstrate the roles of HDACs in intestine (56), oligodendrocyte (40, 53), neuron (25), adipocyte (63), osteoblast (37) and T cell (55) differentiation programs and are now being clinically evaluated as cancer drugs (45). However, to date, pancreatic phenotypes have not been reported in specific HDAC-deficient mice.

SUMMARY OF THE INVENTION

The present invention provides a method for obtaining a population of insulin producing-beta cells which comprises a step of contacting a Pdx1-expressing pancreatic explant with an amount at least one histone deacetylase inhibitor (HDACi). In such a method, the HDACi is selected from the group consisting of selective class II HDACi and classical HDACi. In preferred embodiments, the HDACi is trichostatin A (TSA).

In another aspect, the invention also provides a method for obtaining a population of insulin producing-beta cells which comprises a step of contacting Ngn3-expressing cells with an amount at least one HDACi.

In another aspect, the invention also provides a method for obtaining a population of Ngn3-expressing cells which comprises a step of contacting a Pdx1-expressing pancreatic explant with an amount at least one HDACi. In such a method, the HDACi is selected from the group consisting of selective class I HDACi, selective class II HDACi and classical HDACi. In preferred embodiments, the HDACi is valproic acid (VPA) or TSA.

In yet another aspect, the invention provides a pharmaceutical composition for the treatment of diabetes which comprises an amount of at least one HDACi. The suitable HDACi is selected from the group consisting of selective class II HDACi and classical HDACi. In a preferred embodiment, the HDACi is TSA.

DETAILED DESCRIPTION OF THE INVENTION

The present invention encompasses the recognition by the inventors that the maintenance of acetylation (via histone deacetylase inhibition) has a specific, dominant function in pancreatic lineage development. The inventors have indeed highlighted the ability of histone deacetylase inhibitors to modulate pancreatic cell determination and amplify specific cellular sub-types. Such approach may thus be very useful for developing novel cell replacement therapies in diabetes.

Indeed, the present inventors have now demonstrated that HDACi treatment promoted the Ngn3 pro-endocrine lineage leading to an increased pool of endocrine progenitors and insulin producing-beta cells with potential applications in cell replacement therapies notably in diabetes.

Definitions:

As used herein, the term “pancreatic explant” generally refers to a tissue harvested after its isolation from a piece of pancreas and placed in a culture dish containing an appropriate culture medium.

As used herein, the term “Pdx1-expressing pancreatic explants” means that pancreatic explants which are used in the context of the invention express the marker Pdx1 which is the transcription factor pancreatic duodenal homeobox-1. Therefore, Pdx1-expressing pancreatic explants include embryonic pancreas explants such as the established rat embryonic pancreas explants as previously described (2, 21). Furthermore, Pdx1-expressing pancreatic explants do not produce insulin (at least no detectable amount of insulin). Pdx1-expressing pancreatic explants do not contain insulin producing-beta cells. Thus, the unique type of cell expressing Pdx1 present in such pancreatic explants is pancreatic progenitors.

As used herein, the term “marker” refers to a protein, glycoprotein, or other molecule expressed on the surface of a cell or into a cell, and which can be used to help identify the cell. A marker can generally be detected by conventional methods. Specific, non-limiting examples of methods that can be used for the detection of a cell surface marker are immunohistochemistry, fluorescence activated cell sorting (FACS), and enzymatic analysis.

As used herein, the term “providing” refers to a process in which tissue or organ are isolated and provided in a state suitable for in vitro culture.

As used herein, the term “isolated” refers to a tissue or organ which has been separated from at least some components of its natural environment. This term includes gross physical separation of the tissue or organ from natural environment (e.g., removal from the donor).

As intended herein, the term “contacting” refers to a process in which pancreatic explants are put in contact in an appropriate culture medium with an amount of at least of HDACi of interest.

As used herein, the term “an appropriate culture medium” refers to a culture medium that contains nutrients necessary to support the growth and/or survival of the pancreatic explants and the Ngn3-expressing cells and insulin producing beta-cells. Said appropriate culture medium may or may not further comprise growth factors. By way of example, growth factors of interest may be serum, fibroblast growth factors and any combination of these or other growth factors.

The term “progenitor cells” refers to cells arc partially specialized. These cells divide and give rise to differentiated cells. Progenitor cells belong to a transitory amplifying population of cells derived from stem cells. Compared to stem cells, they have a limited capacity for self-renewal and differentiation. Moreover, since progenitor cells are committed to a particular differentiation process, progenitor cells also express specific markers.

The terms “Ngn3-expressing cells”, “Ngn3+ cells”, “pancreatic endocrine progenitor cells”, insulin producing-beta cell progenitor” or “islet progenitors” are used herein interchangeably. They refer to progenitor cells that can divide and give rise to the different pancreatic endocrine cells (i.e. α cells, β cells, δ cells and PP cells). Such progenitor cells express the specific marker Ngn3 which is a transcription factor which is a marker of choice for detecting the onset of pancreatic endocrine cell differentiation. Ngn3 thus represents the earliest pancreatic endocrine-cell-specific transcription factor in embryonic development and is a well-established marker for embryonic insulin producing-beta cells progenitors. Moreover, such Ngn3-expressing cells express other specific markers such as Pdx1, Pax4, Isl1, Nkx2.2, Nkx6.1, Isl1, NeuroD1 and Pax6 such as described by Chiang et al. 2003 (68).

The terms “beta cells”, “β cells” or “insulin producing-beta cells” are used herein interchangeably. They refer to a type of cell present in the pancreas in areas called the islets of Langerhans. They make up 65-80% of the cells in the islets. Beta cells make and release insulin, a hormone that controls the level of glucose in the blood.

The terms “histone deacetylases” or “HDACs” are used herein interchangeably. They refer to enzymes that remove acetyl groups from histones.

As previously described, mammalian HDACs are grouped into the classical class I, II and IV HDAC family and the structurally unrelated Sirtuin family (class III HDACs). Thus, class I HDACs includes HDAC1-3 and 8, class II HDACs includes HDAC4-7, 9 and 10 and class IV includes HDAC11.

The term “histone deacetylase inhibitor” or “HDACi” are used herein interchangeably. They refer to a compound natural or not which inhibits the histone deacetylase activity. There exist different classes of HDACi in function of their selectivity for their substrates. The classes of HDACi useful in the present invention are selective class I HDACi, selective class II HDACi and classical HDACi.

A “classical HDACi” refers thus to a compound natural or not which has the capability to inhibit the histone deacetylase activity independently of the class of HDACs. Therefore a classical HDACi is a non selective HDACi. By “non selective” it is meant that said compound inhibits the activity of classical HDACs (i.e. class I, II and IV) with a similar efficiency independently of the class of HDAC. Examples of classical HDACi include, but are not limited to, trichostatin A (TSA), SAHA, scriptaid, oxamflatin, NVP-LAQ824, PDX101, LBH-589, ITF2357 and PCI-24781; cyclic peptides such as trapoxine and apicidin; sodium butyrate; benzamides and 2′-amino-anilides such as acetyldinaline and histacin.

In the context of the invention, “selective class I HDACi” is selective for class I HDACs (i.e. HDAC1-3 and 8) as compared with class II HDACs (i.e. HDAC4-7, 9 and 10). By “selective” it is meant that selective class I HDACi inhibits class I HDACs at least 5-fold, preferably 10-fold, more preferably 25-fold, still preferably 100-fold higher than class II HDACs. Selectivity of HDACi for class I or class II HDACs may be determined according to previously described method (Kahn et al. 2008). Examples of selective class I HDACi include, but are not limited to, valproic acid (VPA), sodium valproate, the cyclic peptide FK-228 and 2′-amino-anilides such as MS275, CI994 and MGCD0103.

In the same way, “selective class II HDACi” is selective for class II HDACs (i.e. HDAC4-7, 9 and 10) as compared with class I HDACs (i.e. HDAC1-3 and 8). By “selective” it is meant that selective class II HDACi inhibits class II HDACs at least 5-fold, preferably 10-fold, more preferably 25-fold, still preferably 100-fold higher than class I HDACs. Examples of selective class II HDACi include, but are not limited to, tubacin and (aryloxopropenyl)pyrrolyl hydroxamates.

In the context of the invention, the term “treating” or “treatment”, as used herein, refers to a method that is aimed at delaying or preventing the onset of a pathology, at reversing, alleviating, inhibiting, slowing down or stopping the progression, aggravation or deterioration of the symptoms of the pathology, at bringing about ameliorations of the symptoms of the pathology, and/or at curing the pathology.

As used herein, the term “subject” refers to a mammal, preferably a human being, that can suffer from a condition associated with tissue or organ damage, but may or may not have the pathology. The term “subject” does not denote a particular age, and thus encompasses adults, children, and newborns.

As used herein, the term “amount” refers to any amount of at least HDACi (or a pharmaceutical composition thereof) that is sufficient to achieve the intended purpose.

As used herein, the term “pathologies” refers to any disease or condition associated with tissue or organ damage. The term “pathology associated with tissue or organ damage” refers to any disease or clinical condition characterized by tissue or organ damage, injury, dysfunction, defect, or abnormality. Thus, the term encompasses, for example, injuries, degenerative diseases and genetic diseases. Such pathologies may affect any tissues or organs.

Methods for Obtaining Ngn3-Expressing Cells and Insulin Producing-Beta Cells

In a first aspect, the invention relates to a method for obtaining a population of Ngn3-expressing cells which comprises a step of contacting Pdx1-expressing pancreatic explants with at least one histone deacetylase inhibitor (HDACi).

In a particular embodiment, the method comprises the following steps:

-   -   a) providing a Pdx1-expressing pancreatic explant, and     -   b) contacting said explant with an amount of at least one HDACi.

In such method, the HDACi is selected from the group consisting of selective class I HDACi, selective class II HDACi and classical HDACi.

In an embodiment, the HDACi is valproic acid (VPA).

In another embodiment, the HDACi is trichostatin A (TSA).

In an attempt to overcome the problems of scarcity of insulin producing-beta cells described in the state of the art, the present inventors have developed a simple method for obtaining insulin producing-beta cells.

Thus, in a second aspect the invention relates to a method for obtaining a population of insulin producing-beta cells which comprises a step of contacting a Pdx1-expressing pancreatic explant with an amount at least one HDACi.

In a particular embodiment, the method comprises the following steps:

-   -   a) providing a Pdx1-expressing pancreas explant, and     -   b) contacting said pancreas explant with an amount of at least         one HDACi.

In such method, the HDACi is selected from the group consisting of selective class II HDACi and classical HDACi.

In a particular embodiment, the HDACi is TSA.

In a third aspect, the invention relates to a method for obtaining a population of insulin producing-beta cells which comprises a step of contacting Ngn3-expressing cells with an amount at least one HDACi.

In a particular embodiment, the method comprises the following steps:

-   -   a) providing a population of Ngn3-expressing cells, and     -   b) culturing said population with an amount of at least one         HDACi.

In such method, the HDACi is selected from the group consisting of class II HDACi and classical HDACi.

In a particular embodiment, the HDACi is TSA.

The inventive method thus provides a valuable alternative to obtain a high number of insulin producing-beta cells that can be used as pharmacological and/or therapeutic tools.

Thus, a first step consists in providing a pancreas explant. As already mentioned above, pancreatic explants useful in a method according to the present invention express the transcription factor Pdx1.

In one embodiment, the Pdx1-expressing pancreatic explant is a non-human embryonic pancreatic explant.

In another embodiment, the Pdx1-expressing pancreatic explant is a human foetal pancreatic explant.

Indeed, within the context of the invention, Pdx1-expressing pancreas explants may be from any appropriate mammal origin (e.g., mouse, rat, rabbit, pig, dog or human origin).

The Pdx1-expressing pancreatic explants of the present invention include, but are not limited to, embryonic rat pancreas as previously described (2, 21) or foetal human pancreas as previously described (67). As used herein, the term “foetal” refers to a human developing organism from the eighth week after fertilization.

Embryonic rat pancreas explant may be obtained by dissecting pancreatic epithelia after harvesting embryos at E13.5. The stomach, the pancreas, and a small portion of the intestine were dissected together; then the pancreatic primordium was dissected. It must be noted that at E13.5, the pancreas is composed of an epithelium surrounded by mesenchymal tissue and that in the presence of mesenchyme, the epithelium grew rapidly, spread into the mesenchyme, and developed lobules. It must be further noted that at this stage E13.5, the pancreatic epithelium is mainly composed of early Pdx1 expressing-progenitors, with few glucagon expressing-endocrine cells.

It must also be noted that Pdx1-expressing pancreatic explants may also be characterized by other additional markers such as Pft1a and NKx6.1 (29).

In another embodiment, pancreas explants may be explants harvested from human foetuses (67) from the eighth week after fertilization.

Methods of harvesting samples from tissues and organs are known in the art and can be used in the practice of the present invention. Preferably, methods of harvesting are not excessively destructive for the tissue being harvested. Isolation of samples of interest from a tissue sample preferably occurs in an aseptic environment. For example, the tissue sample may be washed with a buffer solution (e.g., buffered saline) optionally comprising antimytotic and/or antibiotic agents.

A second step consists in contacting a Pdx1-expressing pancreas explant or a population of Ngn3-expressing cells with an amount of at least HDACi in an appropriate culture medium.

An appropriate culture medium according to the invention may consist in a minimal medium in which cells can grow, such as for example an RPMI medium complemented with serum. Such medium may also include others factors of interest such antibiotics or amino acids. A defined medium consisting of RPMI 1640 supplemented with penicillin (100 units/ml), streptomycin (100 μg/ml), HEPES (10 mmol/l), L-glutamine (2 mmol/l), nonessential amino acids (1x), and 10% heat-inactivated calf serum may be typically used as appropriate culture medium.

In certain embodiments, the culture medium is changed every day.

Cultures are often grown in a suitable vessel in a sterile environment at 37° C. in an incubator containing a humidified 95% air—5% CO2 atmosphere. Vessels may contain stirred or stationary cultures. Cell culture techniques are well known in the art and established protocols are available for the culture of diverse cell types.

Pdx1-expressing pancreatic explants may be put in contact with at least appropriate HDACi for any efficient amount of time, i.e., any amount of time that is necessary to allow the formation of pancreatic endocrine progenitor cells or insulin producing-beta cells. One skilled in the art will know how to determine such an amount of time.

In embodiments destined for obtaining a population of Ngn3-expressing cells, Pdx1-expressing pancreatic explants are put in contact with at least one HDACi for at least about 3 days, preferably between 3 days and more preferably for 7 days in an appropriate culture medium. In embodiments destined for obtaining insulin producing-beta cells, Pdx1-expressing pancreatic explants are put in contact for at least about 7 days and preferably for at least about 15 days in an appropriate culture medium with at least one HDACi.

Moreover, Pdx1-expressing pancreatic explants or Ngn3-expressing cells may be put in contact with at least one appropriate HDACi in any efficient amount, i.e., any amount that is necessary to allow the formation of Ngn3-expressing cells or insulin producing-beta cells. One skilled in the art will know how to determine such an amount. It must be underlined that useful HDACi concentration depends on kind of used pancreatic explant (e.g. higher for human explant than rat explant).

Thus, in a method according to the present invention, the concentration of VPA added in the culture medium is preferably comprised between about 1 mM and about 3 mM. In another method according to the present invention, the concentration of TSA added in the culture medium is preferably comprised between about 70 nM and about 700 nM.

In a preferred embodiment involving embryonic rat pancreas explant, the optimal concentration of VPA added in the culture medium is about 1 mM. In another preferred embodiment, the optimal concentration of TSA added in the culture medium is about 100 nM.

A large number of known HDACi can be used in the practice of the invention.

The selective class I HDACi of the present invention include, but are not limited to, valproic acid (VPA), sodium valproate; the cyclic peptide FK-228 and 2′-amino-anilides such as MS275, CI994 and MGCD0103.

The selective class II HDACi of the present invention include, but are not limited to, tubacin and (aryloxopropenyl)pyrrolyl hydroxamates.

The classical HDACi of the present invention include, but are not limited to, hydroxamates such as trichostatin A (TSA), SAHA, scriptaid, oxamflatin, NVP-LAQ824, PDX101, LBH-589, ITF2357 and PCI-24781; cyclic peptides such as trapoxine and apicidin; sodium butyrate (NaB); benzamides and 2′-amino-anilides such as acetyldinaline and histacin.

Cells of interest obtained with a method according to the present invention may also be expanded by culturing in an appropriate culture medium comprising at least one factor that stimulates the proliferation of the cells.

Thus, a method according to the present invention constitutes a quick and easy way to obtain a high number of Ngn3-expressing cells and insulin producing-beta cells that can be used in therapeutic applications.

It must further be noted that the population of Ngn3-expressing cells or the population of insulin producing-beta cells that are obtained by any method disclosed herein may be incorporated in a pharmaceutical composition, optionally with a pharmaceutically acceptable carrier or excipient. Thus, a population of pancreatic endocrine progenitor cells or a population of insulin producing-beta cells obtained by a method of the present invention, or a pharmaceutical composition thereof, may be used in the treatment of pathologies, in particular pathologies associated with pancreas damage, injury, dysfunction, degeneration or abnormality such as diabetes, and for reconstruction or regeneration of the pancreatic endocrine tissue.

Pharmaceutical Composition for the Treatment of Diabetes which Comprises an Amount of at least one HDACi.

A further aspect of the invention relates to pharmaceutical composition for the treatment of diabetes which comprises an amount of at least one HDACi.

Such pharmaceutical composition may be useful for inducing in a subject the differentiation of endogenous progenitors in insulin producing-beta cells.

In such pharmaceutical composition, the HDACi is selected from the group consisting of class II HDACi and classical HDACi.

In a particular embodiment, the HDACi is TSA.

In certain embodiments, a pharmaceutical composition may further comprise at least another biologically active substance or bioactive factor.

As used herein, the term “pharmaceutically acceptable carrier or excipient” refers to a carrier medium which does not interfere with the effectiveness of the biological activity of the HDACi, and which is not excessively toxic to the host at the concentrations at which it is administered. Examples of suitable pharmaceutically acceptable carriers or excipients include, but are not limited to, water, salt solution (e.g., Ringer's solution), alcohols, oils, gelatins, carbohydrates (e.g., lactose, amylase or starch), fatty acid esters, hydroxymethylcellulose, and polyvinyl pyroline. Pharmaceutical compositions may be formulated as liquids, semi-liquids (e.g., gels) or solids (e.g., matrix, lattices, scaffolds, and the like). If desired, the pharmaceutical composition may be sterilized.

As used herein the term “biologically active substance or bioactive factor” refers to any molecule or compound whose presence in a pharmaceutical composition of the invention is beneficial to the subject receiving the composition. As will be acknowledged by one skilled in the art, biologically active substances or bioactive factors suitable for use in the practice of the present invention may be found in a wide variety of families of bioactive molecules and compounds. For example, a biologically active substance or bioactive factor useful in the context of the present invention may be selected from anti-inflammatory agents, anti-apoptotic agents, immunosuppressive or immunomodulatory agents, antioxidants, growth factors, and drugs.

A related aspect of the invention relates to a method for treating a subject suffering from a pathology associated with tissue or organ damage, said method comprising a step of administering to the subject an efficient amount of HDACi as described herein, or a pharmaceutical composition thereof.

Another aspect of the invention relates to a method for treating a subject suffering from a pathology associated with tissue or organ damage, said method comprising a step of administering to the subject a population of insulin producing-beta cells as obtained by a method according to the present invention, or a phaimaceutical composition thereof.

In a particular embodiment, the method comprises the following steps:

-   -   a) providing a population of insulin producing-beta cells, and     -   b) administrating said population to a subject in need thereof.

Still another aspect of the invention relates to a method for treating a subject suffering from a pathology associated with tissue or organ damage, said method comprising a step of administering to the subject a population of Ngn3-expressing cells as obtained by a method according to the present invention, or a pharmaceutical composition thereof.

In a particular embodiment, the method comprises the following steps:

-   -   a) providing a population of Ngn3-expressing cells, and     -   b) administrating said population to a subject in need thereof.

Yet, in preferred embodiments, pathology of interest is diabetes mellitus. Diabetes mellitus often simply diabetes is a syndrome characterized by disordered metabolism and inappropriately high blood sugar (hyperglycaemia) resulting from either low levels of the hormone insulin or from abnormal resistance to insulin's effects coupled with inadequate levels of insulin secretion to compensate. The World Health Organization recognizes three main forms of diabetes mellitus: type 1, type 2, and gestational diabetes (occurring during pregnancy), which have different causes and population distributions. While, ultimately, all forms are due to the beta cells of the pancreas being unable to produce sufficient insulin to prevent hyperglycemia, the causes are different. Type 1 diabetes is usually due to autoimmune destruction of the pancreatic beta cells. Type 2 diabetes is characterized by insulin resistance in target tissues. This causes a need for abnormally high amounts of insulin and diabetes develops when the beta cells cannot meet this demand. Gestational diabetes is similar to type 2 diabetes in that it involves insulin resistance; the hormones of pregnancy can cause insulin resistance in women genetically predisposed to developing this condition.

Effective dosages and administration regimens can be readily determined by good medical practice based on the nature of the pathology of the subject, and will depend on a number of factors including, but not limited to, the extent of the symptoms of the pathology and extent of damage or degeneration of the tissue or organ of interest, and characteristics of the subject (e.g., age, body weight, gender, general health, and the like).

The invention will be further illustrated by the following figures and examples. However, these examples and figures should not be interpreted in any way as limiting the scope of the present invention.

FIGURES

FIG. 1 is a schematic representation of pancreatic differentiation.

During pancreatic development, precursor cells expressing the PDX1-homeodomain transcription factor give rise to both exocrine and endocrine pancreatic cell lineages. Mature exocrine acinar cells express digestive enzymes such as amylase. The endocrine cell sub-types including insulin-expressing β-cells as well as α, δ and PP cells, arise from endocrine progenitors expressing the NGN3 transcription factor.

The morphology of a E13.5 rat pancreatic explant cultured 1 day is shown, with the epithelium circled. Immunohistological analysis shows PDX1 staining in epithelial cells (in green). Ngn3 transcripts (in blue) are revealed by in situ hybridization in the pancreatic explant cultured 5 days. Exocrine acinar cells are detected by amylase staining (in green), and endocrine insulin-expressing cells are detected by insulin staining (in red), with immunohistological analysis of pancreatic explants cultured 7 days.

FIG. 2 is a schematic representation of pancreatic cell lineage differentiation. Pdx1 expressing-progenitor cells give rise to both exocrine and endocrine cells. Among others, the transcription factor P48 is involved in the commitment of the acinar exocrine cells. Endocrine progenitors express the transcription factor NGN3. Downstream of Ngn3, the transcription factors Arx and Pax4 among others, are involved in the determination of the different endocrine sub-types.

FIG. 3 shows that HDACi treatment enhances the pool of Ngn3 endocrine progenitor cells.

FIG. 5A is a graph representing real-time PCR quantification of Ngn3 mRNA after 0, 1, 3, 5, 7, 9, 11 and 14 days of culture, with and without VPA or TSA treatment. Values are meansSEM of at least three independent experiments; **p<0.005; ***p<0.001.

FIG. 5B is a set of pictures showing at top the detection of Ngn3 transcripts by in situ hybridization in pancreases cultured for 5 days, with and without VPA or TSA treatment; and at bottom the detection of Ngn3 protein by immunohistochemistry in pancreases cultured 7 days, with and without VPA or TSA treatment. Scale bar=100 μm.

FIG. 4 shows the opposite effects of VPA and TSA treatment on the endocrine β/δ lineage differentiation.

FIG. 4A is a set of pictures representing pancreases cultured 9, 11 and 14 days with and without VPA or TSA treatment. Note that with TSA treatment, at day 11 and 14, translucent buds can be seen (black arrows).

FIG. 4B is a set of graph representing real-time PCR quantification of insulin mRNA after 7, 9, 11 and 14 days of culture, with and without VPA or TSA treatment.

FIG. 4C is a set of pictures illustrating the mmunohistological analyses of pancreases after 14 days of culture, with and without VPA or TSA treatment. β-cell development was evaluated using anti-insulin staining, in red. Nuclei were stained in blue with Hoechst. Note that in TSA-treated explants, insulin staining (white arrow) corresponds to the translucent bud seen in A (black arrow). Quantification of the absolute surface areas occupied by insulin+ cells that developed after 14 days of culture, without or with VPA or TSA treatment. Values are meansSEM of at least three independent experiments. NS: no significant difference; *p<0.05; **p<0.005; ***p<0.001. Scale bar=100 μm.

FIG. 5 is a schematic representation of class I HDAC inhibitors effects on pancreas differentiation.

VPA and MS275 (class I HDAC inhibitors) repress acinar lineage to the benefit of ductal cell differentiation. These HDACi promote endocrine NGN3 progenitor cells. The differentiation of α and PP cells is enhanced while the β/δ lineage is abolished. More glucagon- and PP-expressing endocrine cells are thus induced by VPA and MS275.

FIG. 6 is a schematic representation of class I and class II HDAC inhibitors effects on pancreas differentiation.

TSA and NaB (both class I and class II HDAC inhibitors) repress acinar lineage to the benefit of ductal cell differentiation. These HDACi promote endocrine NGN3 progenitors cells that differentiate into the α/PP and β/δ lineages. More endocrine cells, especially insulin-expressing δ cells, are thus generated by TSA and NaB.

FIG. 7 represents a model for the role of HDACs on pancreas differentiation.

Simple and double lines delineate the proposed involvement of class I and class II HDACs, respectively, in the regulation of pancreatic lineages. Based on the effects of HDACi, we propose that class I HDACs regulate the exocrine lineage commitment in acinar and ductal cells. We propose that class I HDACs have a major effect on controlling the NGN3 pro-endocrine lineage. We propose that class I and class II HDACs have distinct role in the regulation of endocrine sub-types. Class I and class II HDACs might regulate the balance between Pax4 and Arx expression, two transcription factors involved in the β/δ and α/PP lineages respectively. According to our model, when class I HDACs are inhibited (by VPA or MS275) Arx is promoted, favoring Pax4 inhibition and repression of the β/δ lineage. When all HDACs are repressed (by TSA or NaB), Arx and Pax4 are enhanced and both β/δ and α/PP lineages are induced.

EXAMPLE 1 Material & Methods

Animals and dissection of dorsal pancreatic rudiments: Pregnant Wistar rats were purchased from the CERJ (Le Genest, France). The first day post-coitum was taken as embryonic day 0.5 (E0.5). Pregnant female rats at 13.5 days of gestation were killed by CO2asphyxiation, according to the French Animal Care Committee's guidelines. Dorsal pancreatic buds from E13.5 rat embryos were dissected as described previously (2).

Organ culture, HDACi treatments, BrdU incorporation: Pancreases were laid on 0.45 μm filters (Millipore) at the air-medium interface in Petri dishes, containing RPMI 1640

(Invitrogen) supplemented with penicillin (100 U/mL), streptomycin (100 μg/mL), HEPES (10 mmol/L), L-glutamine (2mmol/L), non-essential amino acids (1x, Invitrogen) and 10% heat-inactivated calf serum (HyClone). Cultures were maintained at 37° C. in humidified 95% air/5% CO2. Medium was changed every other day. Explants were cultured in the presence of VPA or TSA (Sigma). We determined the optimal concentration of both inhibitors by testing increasing doses of VPA (from 0.75 mM to 3 mM) and TSA (from 50 nM to 200 nM). Because VPA concentrations above 1.5 mM and TSA concentrations above 125 nM result in increased cell death (data not shown), we used concentrations that lead to phenotypic effects without toxicity: 1 mM VPA and 100 nM TSA. MS275 and NaB (Sigma) were used at 1 μM and 125 μM, respectively. For cell proliferation assay, 10 μM BrdU (Sigma) was added to the medium during the last hour of culture.

Immunohistochemistry and quantification: Tissues were fixed in 10% formalin, pre-embedded in low gelling agarose and embedded in paraffin. All sections (4 μm thick) of each pancreatic explant were collected and processed for immunohistochemistry, as described previously (14, 46). Antibodies were used at the following dilutions: mouse anti-insulin (Sigma, 1/2000), guinea pig anti-insulin (DAKO, 1/200), mouse anti-glucagon (Sigma, 1/2000), rabbit anti-amylase (Sigma, 1/300), goat anti-osteopontin/SPP1 (R&D systems, 1/200), rabbit anti-PDXI ((14), 1/1000), mouse anti-BrdU (Amersham, ½), rabbit anti-Ngn3 ((21), 1/1000). The fluorescent secondary antibodies were: fluorescein anti-rabbit and anti-goat antibodies (Jackson Immunoresearch, 1/200), Texas-red anti-mouse antibody (Jackson Immunoresearch, 1/200) Alexa fluor 488 anti-rabbit antibody (Biogenex, 1/400) and AMCA anti-guinea pig antibody (Jackson Immunoresearch, 1/200). Nuclei were stained blue with Hoechst 33342 (0.3 μg/ml, Invitrogen). Ngn3 detection was perfointed as previously described (21) using the Vectastain elite ABC kit (Vector Laboratories). Photographs were taken using a fluorescence microscope (Leitz DMRB, Leica) and digitized using cooled 3CCD cameras (C5810 or C7780, Hamamatsu). The surface area of each staining was quantified with IPLab (Scanalytics). The surface areas per section were summed to obtain the total surface area per explant in mm2. At least three explants were analyzed per condition and results are expressed as means±SEM. Statistical significance was determined using Student's t test.

TUNEL labelling: Terminal deoxynucleotidyltransferase-mediated dUTP-biotin nick end labeling (TUNEL) experiments were performed using an in situ cell death detection kit (Roche) and followed by insulin and amylase immunostaining.

In situ hybridization: In situ hybridization was performed as previously described (15) and colorimetric development was performed with 5-bromo-4-chloro-3-indolyl phosphate (Promega) and nitroblue tetrazolium (Roche). No signal was obtained when a sense riboprobe was used.

RNA extraction and real-time PCR: Total RNA was extracted from pools of at least three pancreases using RNeasy Microkit (Qiagen) and reverse transcribed using Superscript reagents (Invitrogen). Real-time PCR was performed with the 7300 Fast real-time PCR System; each reaction consisted of a mix of Taqman® universal PCR master mix with a specific labelled probe (Applied Biosystem) (21). The comparative method of relative quantification (2^(−ΔΔct)) (38) was used to calculate expression levels of each target gene, normalized to peptidylpropyl isomerase A (ppia/cyclophilin A). The data are presented as fold changes in gene expression. At least three pools of explants were analyzed per condition and results are expressed as means±SEM. Statistical significance was determined using Student's t test. PCR primer sequences are available upon request.

Protein extracts and Western-Blot analysis: Tissue lysates were prepared from pools of at least five pancreases using a Complete Lysis-M kit (Roche). Equal amounts of proteins were loaded on SDS-PAGE gels for separation and transferred onto 0.45 μm nitrocellulose membranes. After blocking with milk, the membranes were probed with different antibodies: rabbit anti-HDAC1, rabbit anti-HDAC2, rabbit anti-HDAC3, rabbit anti-HDAC4, rabbit anti-HDAC6, rabbit anti-HDAC7 (Abeam) and rabbit anti-HDAC5 (Sigma), rabbit anti-acetyl-histone H3 (Lys9), rabbit anti-acetyl-histone H4 (Lys12) (Upstate) and mouse anti-actin antibody (Sigma). Results were visualized with horseradish peroxidase-conjugated secondary antibodies (Cell Signaling) and enhanced chemiluminescence (LumiGLO, Cell Signaling).

HDAC enzyme activity assay: Total cellular HDAC enzyme activity was measured using an HDAC assay kit (Millipore). Briefly, 2 μg of control or VPA- or TSA-treated cell extracts (prepared as described above) were incubated in a 96-well plate with a fluorometric substrate in HDAC assay buffer for 45 min at 37° C. An activator solution was then added to release the fluorophore from the deacetylated substrates, and fluorescence was measured in a plate-reading fluorimeter (excitation=390 nm, emission detection=460 nm).

EXAMPLE 2

In vivo HDACi injections in rat: Experiments were submitted for ethical evaluation to the “Comité Régional d′Ethique pour l′Experimentation Animale Ile-de-France-Paris Descartes” and approved (register number P2.CHS.081.09). Pregnant rat wistar females are injected intraperitoneally daily with HDACi between E13.5 and E20.5. The NaB treated group received 1000 mg/kg whereas the VPA treated group received 300 mg/kg, in Phosphate Buffer Saline solution (PBS). After 7 days of treatment, the animals are killed by CO2 asphyxiation, according to the French Animal Care Committee's guidelines. Embryos are dissected and each pancreas is fixed or frozen for subsequent analysis by immunohistochemistry or western-blot respectively.

EXAMPLE 3

In vitro HDACi treatment of human pancreas: Human pancreases are extracted from foetal tissue fragments obtained immediately after voluntary abortions performed around 9 weeks of development, in compliance with the current French legislation. Human pancreases are cultured in the same conditions described above used for rat pancreases, and treated until 14 days with 800nM TSA.

Results

HDACs are down-regulated during pancreas development: The inventors first analyzed the expression of different HDACs during rat pancreas development. Using Western blotting, we found that both class I (HDAC1-3) and class II (HDAC4-7) HDACs were expressed at E13.5, E17.5 and in the adult pancreas. The expression levels of most HDACs (with the exception of HDAC3) decreased during development. The inventors next measured total HDAC activity and observed a 86.1% ±6.5% decrease at E17.5 compared with E13.5. To determine whether decreased HDAC activity correlated with increased histone acetylation, they performed Western blot analysis with antibodies directed against histone acetyl-H3 and acetyl-H4 residues. The inventors found that the overall degree of histone acetylation increased from E13.5 to the adult. These results show that HDACs are expressed and then developmentally regulated in the pancreas. Decreased HDAC expression is associated with decreased HDAC activity and increased histone acetylation throughout pancreas development.

To study the role of HDACs in pancreas development, the inventors treated rat pancreatic explants with VPA and TSA. They cultured E13.5 rat pancreases on floating filters at the air-medium interface for 14 days. Under such conditions, acinar and endocrine cells develop in a way that replicates pancreas development in vivo (2, 21). The inventors found that the in vitro HDAC expression pattern replicates the one found in vivo, with decreased expression of most HDACs. They next measured total HDAC activity from day 0 to day 14 and found a 65.4%±1.6% decrease between the two stages. They verified that VPA and TSA were acting as HDACi, and found average decreases of 93.2%±2.3% and 99.2%±0.9% with VPA and TSA, respectively. The decreased HDAC activity was correlated with an increase in histone acetylation by Western Blot analysis with histone acetyl-H3 and -H4 antibodies and found that both HDACi induced histone hyper-acetylation. This correlated with 93.2%±2.3% and 99.2%±0.9% decreases in HDAC activity with VPA and TSA, respectively. Moreover, they found that both HDACi induced histone hyperacetylation.

Altogether, these results show that HDAC expression and activity are regulated during pancreas development in vivo and in vitro. Thus, data validate the use of VPA and TSA for potently modulating HDAC activity in pancreatic explants.

HDACi treatment suppresses acinar differentiation and promotes ductal differentiation: The inventors first compared the morphology of pancreases cultured in the presence or absence of HDACi. At E13.5, the pancreas is composed of an epithelium surrounded by mesenchymal tissue. In control conditions, the epithelium grew and spread into the mesenchyme. With VPA or TSA treatment, epithelial growth occurred, branching increased and, after 7 days of culture, the tips of the branched epithelium formed cystic structures. Haematoxylin/eosin staining revealed cystic structures and less-developed acinar structures with VPA and TSA, compared with controls. They found similar results with MS275 and NaB.

To precisely define the molecular events resulting from HDAC inhibition, the inventors analyzed markers for the different pancreatic lineages. They found no effect of HDACi on the ratio of epithelial to mesenchymal tissue (by analyzing E-cadherin and vimentin expression, respectively; data not shown). They next analyzed the effects of HDACi on cell differentiation, focusing first on the exocrine lineage. They used real-time PCR to monitor the expression of the P48/Ptfla (33) and Mist1 (51) transcription factors involved in acinar cell development, and amylase, a marker of differentiated acinar cells. In control conditions, the expression of P48 and Mist1 increased over the first 5 days of culture, followed by increased amylase expression. The increase in levels of P48, Mist1 and amylase was strongly reduced by VPA or TSA. Quantification of the surface area occupied by amylase+ cells indicated that acinar cell development was much lower in pancreases cultured with VPA or TSA. For ductal cell differentiation, the inventors used real-time PCR to evidence a major increase in SPP1 (31) expression, especially after 5 and 7 days of culture with VPA or TSA. Immunohistological analysis showed that SPP1 staining was greater in HDACi-treated pancreases and was mainly found around cystic structures. Quantification of the surface area occupied by SPP1+ cells showed a 4-fold increase on HDACi treatment, indicating enhanced ductal cell development. Similar results with exocrine differentiation were found with MS275 and NaB.

Overall, these results show that HDACi treatment leads to a dramatic decrease in acinar lineage differentiation, whereas ductal lineage differentiation is strongly enhanced.

HDACi treatment strongly promotes Ngn3 pro-endocrine lineage: To define whether HDAC activity was involved in the regulation of endocrine lineage, the inventors focused on expression of Ngn3, a specific pancreatic endocrine progenitor marker (18). In control pancreatic explants, Ngn3 mRNA levels measured by real-time PCR increased after 1 day of culture, peaked at day3 and decreased thereafter. With VPA or TSA, the inventors observed a dramatic increase in Ngn3 expression from day1 and a peak at day7 (14-fold higher than in controls). Hence, the Ngn3 expression profile was longer and amplified with HDACi treatment. They found similar results with MS275 and NaB.

At day5, the inventors visualized Ngn3 mRNA expression by in situ hybridization and observed enhanced Ngn3 expression, with positive cells throughout the epithelium in VPA- and TSA-treated explants. Finally, immunohistochemical analysis at day7 showed that NGN3+ cells were extremely scarce in control pancreases, while many were present in VPA- and TSA-treated explants.

Thus, these results show that the expression profile of the pro-endocrine transcription factor Ngn3 was strongly enhanced and maintained with HDACi treatment, leading to an increased pool of endocrine progenitor cells.

HDACi treatment enhances the endocrine α/PP lineage: The inventors next analyzed the effect of HDACi on each of the pancreatic endocrine cell types. They first focused on expression of Arx, known to support an α and PP cell fate (8, 9, 11). They observed a major increase in Arx expression with VPA or TSA treatment at all stages of culture. At day 7, Arx expression was enhanced around 3-fold with VPA and 11-fold with TSA. They next used real-time PCR to monitor the expression of glucagon and PP synthesized by α and PP cells, respectively. Both VPA and TSA treatment produced a major increase in glucagon and PP expression at all stages of culture. At day7, we observed a 5-fold and a 25-fold increase in glucagon expression and a 4-fold and a 6-fold increase in PP expression with VPA and TSA, respectively. Immunohistological analysis revealed a 4-fold increase in the number of glucagon-expressing cells upon VPA or TSA treatment. Similar results were found with MS275 and NaB.

Together, these results show that HDACi treatment promotes a and PP lineage differentiation.

Opposing effects of VPA and TSA on the endocrine β/δ lineage: The inventors used real-time PCR to monitor expression of Pax4, known to support the β/δ cell fate (54) over 7 days of culture. VPA produced a dramatic decrease in Pax4 expression as early as day1, associated with a dramatic decrease in insulin expression and almost full abolition of somatostatin expression. Moreover, the expression of NeuroD1, another Ngn3 target subsequently expressed in δ cells (26, 48) was significantly lower in VPA-treated pancreases. These results were confirmed immunohistologically, with a major decrease in the number of insulin+ cells at days 5 and 7. Even in tissues cultured for 14 days with VPA, insulin expression remained dramatically low. Few insulin+ cells were observed immunohistochemically.

In contrast, TSA strongly activated Pax4 and NeuroD1 expression. However, no differences in insulin and somatostatin expression were found after 7 days in culture either by real-time PCR or by immunohistochemistry. Hence, the inventors extended the culture period. After 11 days, TSA-treated pancreases developed translucent buds at the periphery of the pancreas corresponding to an outer mass of insulin+ cells. Quantification of insulin staining showed that the δ cell mass had increased almost 2-fold under such conditions. Such results were confirmed using real-time PCR: insulin expression was increased 2-fold at day9, 3-fold at day11 and 5-fold at day14 compared with controls. Somatostatin expression was also increased after 14 days in culture (data not shown), demonstrating that the β/δ lineage was promoted by TSA. Interestingly, MS275 showed results similar to those with VPA, whereas NaB showed results similar to those with TSA.

Altogether, these results indicate that VPA suppresses the β/δ lineage, whereas TSA enhances it.

The effects of HDACi treatment on pancreatic differentiation are independent of proliferation and apoptosis: The observed effects of HDACi on pancreatic development could be attributed to a direct effect of HDAC inhibition on the differentiation program itself or to an indirect effect on proliferation/apoptosis. First, the inventors asked whether the massive increase in the number of NGN3+ cells was accompanied by an increased proliferation of early PDX1+ pancreatic precursor cells that would give rise to more NGN3+ endocrine progenitor cells (20). Thus, they cultured pancreases for 1 day with or without VPA and TSA and added BrdU to the medium during the last hour of culture. No difference in BrdU incorporation was seen. The percentage of PDX1+/BrdU+ was 30.4±1.4% in control conditions, 29.3±2.3% with VPA and 31.9±1.4% in TSA-treated pancreases. They next determined whether the greater number of NGN3+ cells was associated with increased proliferation of such cells. After 3 days of culture, very few NGN3/BrdU+ cells could be found in control conditions and neither VPA nor TSA increased the proliferation of NGN3+ cells. Given the observed increase in glucagon-expressing cells with HDACi treatment, the inventors also tested for increased proliferation of such cells. As for NGN3+ cells, they observed a low proliferative rate for glucagon+ cells with no difference between controls and 5-day VPA or TSA. Finally using the TUNEL method, they found that neither VPA nor TSA treatments modified the number of apoptotic cells expressing amylase or insulin, indicating that the lower number of amylase and insulin cells observed with VPA treatment was not due to apoptosis.

These results show that HDACi treatment does not modify the proliferation/apoptosis balance and further suggest that the effects of HDACi treatment on pancreas differentiation are direct.

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1. A method for obtaining a population of insulin producing-beta cells which comprises a step of contacting a Pdx1-expressing pancreatic explant with an amount at least one histone deacetylase inhibitor (HDACi).
 2. The method according to claim 1, wherein the Pdx1-expressing pancreatic explant is a non-human embryonic pancreatic explant or a human foetal pancreatic explant.
 3. A method for obtaining a population of insulin producing-beta cells which comprises a step of contacting Ngn3-expressing cells with an amount at least one HDACi.
 4. The method according to claim 1, wherein the histone deacetylase inhibitor is selected from the group consisting of selective class II HDACi and classical HDACi.
 5. The method according to claim 4, wherein the HDACi is trichostatin A (TSA).
 6. A method for obtaining a population of Ngn3-expressing cells which comprises a step of contacting a Pdx1-expressing pancreatic explant with an amount at least one HDACi.
 7. The method according to claim 6, wherein the HDACi is selected from the group consisting of selective class I HDACi, selective class II HDACi and classical HDACi.
 8. The method according to claim 7, wherein the HDACi is valproic acid (VPA) or TSA.
 9. A pharmaceutical composition for the treatment of diabetes which comprises an amount of at least one HDACi.
 10. The pharmaceutical composition according to claim 9, wherein the HDACi is selected from the group consisting of selective class _11 HDACi and classical HDAC1.
 11. The pharmaceutical composition according to claim 10, wherein the HDACi is TSA. 